一种罕见的boanmycin相关物质的分离纯化和结构测定

从boanmycin产生菌轮枝链霉菌平阳变种(Streptomyces verticillus var. pingyangensis n. sp.)发酵产物中分离、纯化boanmycin相关物质A601。对A601进行光谱分析鉴定其化学结构,结果证实A601的化学结构为博来霉素B6,并对其碳谱进行了归属。

Activity of boanmycin against colorectal cancer

INTRODUCTIONBoanmycin (Bleomycin A6, BAM ), a newantitumor antibiotic, was isolated from manycomponents of bleomycin (BLM) produced bystreptomyces pingyangensis which were obtainedfrom a soil sample collected in Pingyang County,Zhejiang Province, China. Boanmycin has a similarchemical structure to that of BLM, but the terminalamine moiety is different[ 1].

POTENTIATION OF BOANMYCIN ANTITUMOR ACTIVITY BY CHEMOTACTIC PEPTIDE

Objective: Chemotactic peptide may interfere with the process of tumor growth, invasion and metastasis by activating and attracting leukocytes containing macrophages. fMLP (CHO-Met-II e-Phe) is one of the chemotactic peptides. Boanmycin (BAM), a single A6 component from the bleomycin complex, is effective against a panel of cancers in clinical trials. This study was set to investigate the antitumor activity of BAM in combination with chemotactic peptide fMLP. Methods: Cytotoxicity of BAM and fMLP to cancer cells was determined by MTT assay. Therapeutic effect was evaluated by using the model of subcutaneously transplanted hepatoma 22 in mice. Results were judged as that a CDI less than 0.85 was considered as synergism and one less than 0.75 as significant synergism. Results: BAM and fMLP showed no synergism in cytotoxicity to cancer cells. In all in vivo experiments, fMLP was administered peritumorally at the dose of 1 mg/mouse; no significant inhibition by fMLP alone on the growth of hepatoma 22 was found. Different settings of BAM and fMLP combination included: (1) BAM, administered peritumorally×3, was started 24 h after tumor inoculation. BAM (0.5 mg/kg) alone and BAM-fMLP combination inhibited the growth of hepatoma 22 by 26.6% and 64.7%, respectively (P<0.05, CDI=0.36) on day 13. (2) BAM, administered ip×3, was started 24 h after tumor inoculation. The growth of tumor in BAM (1 mg/kg) group was faster than that in BAM-fMLP combination group. On day 14, BAM (1 mg/kg) alone and BAM-fMLP combination suppressed the growth of tumor by 11% and 70.6%, respectively (P<0.05), CDI=0.42). (3) BAM, administered ip×3, was started 96 h after tumor inoculation. The growth of tumor in BAM (1 mg/kg) group was faster than that in BAM-fMLP combination group. On day 13, BAM (1 mg/kg) alone and BAM-fMLP combination suppressed tumor growth by 38.2% and 77.1%, respectively (P<0.05, CDI=0.51). As shown in all in vivo experimental settings, antitumor effect of BAM in combination with fMLP was much more potent than that of BAM alone. Conclusion: This experiment shows that chemotactic peptide fMLP may enhance the antitumor effect of BAM, which indicates that chemotactic modulation may play a positive role in cancer chemotherapy.

Synergistic Action of fMLP-boanmycin Combination on the Growth of Mouse Colon Carcinoma and Its Action Mechanisms

Objective： The inhibitory action of fMLP-boanmycin （BAM） combination on the growth of mouse colon carcinoma and its action mechanisms were observed in order to provide experimental proof for probing novel regimen of chemotactic modulation in combination with chemotherapy in the treatment of cancer. Methods： Cytotoxicity of BAM-fMLP combination to tumor cells was determined by MTT assay in vitro. Antitumor activity of BAM-fMLP combination was assessed in mice subcutaneously transplanted colon carcinoma 26. The amount of superoxide anion （O2 ·^-）released from fMLP stimulated macrophages was determined by NBT assay. The amount of nitric oxide （NO） was indirectly determined by Griess method. Results： BAM-fMLP combination had no synergistic effect on tumor cells（CDI〉0.85）, but BAM at the doses of 10μg/ml, 30μg/ml and 100μg/ml in combination with fMLP at the concentration 20μg / ml exhibited synergistic effect on tumor cells in the presence of macrophages（CDI〈0.75）, fMLP inhibited the growth of colon carcinoma 26 by 50.0% when it at dose of 1 mg/mouse was administered peritumorally. BAM （1 mg/kg, intraperitoneally, three times） alone and BAM - fMLP combination inhibited the growth of colon carcinoma 26 by 38.6% and 78.4%, respectively （CDI=0.71） on day 12. The amount of O2 ·^- released from fMLP 4.6×10^-7 mol/L （0.2μg/ml） stimulated macrophages which were treated by BAM in vitro increased significantly（P〈0.01）, fMLP 2.3×10^-6 mol/L （1μg/ml） could not stimulate macrophages to release NO, but may stimulate macrophages treated with BAM 10μg/ml and 100μg/ml to release NO significantly（P〈0.01）. Conclusion： The inhibitory action of fMLP-boanmycin combination on the growth of mouse colon carcinoma have synergism, which may associate with the increase of O2 ·^- and NO released by macrophages. Chemotactic modulation in combination with chemotherapy may be a novel regimen in the treatment of cancer.

Effect of peritumoral injection of boanmycin hydrochloride within temperature-sensitive in situ gel using Hep-G2 hepatoma nude mice model

Background Boanmycin hydrochloride, a new antitumor agent, has a short half-life and fast clearance speed in vivo. The aim of this research was to investigate the effectiveness of peritumor injection of boanmycin hydrochloride within temperature-sensitive gel in situ using Hep-G2 hepatoma nude mice model. Methods Nude mice with human Hep-G2 tumor in right flank were randomly divided into four groups： normal saline group, in situ gel only group, boanmycin hydrochloride in situ saline group, and boanmycin hydrochloride in situ gel group, and were treated with injection of corresponding agents into peripheral tissue of the tumor. The volume of the tumor and the body weight of the mice were regularly measured, and tumor growth curve was generated. The size, internal echo, and blood flow of the tumors were observed by color Doppler ultrasonography. Histopathologic changes of the tumor after treatment were observed under both optical and transmission electron microscopy. Results The tumor growth was significantly inhibited by peritumoral therapy in boanmycin hydrochloride in situ gel group with the tumor inhibitory rate of 86.76%. The blood flow of the tumor was still seen in both normal saline group and in situ gel only group on color Doppler ultrasound. Punctate calcification and dotted blood flow were seen in boanmycin hydrochloride group; however, there was massive calcification and no blood flow in the tumor in the boanmycin hydrochlodde in situ gel group. Large areas of necrosis and apoptotic cells were shown by microscopic observation in boanmycin hydrochloride in situ gel group. Conclusion Temperature-sensitive boanmycin hydrochloride in situ gel can effectively delay the release of boanmycin hydrochloride and increase its anticancer effects for liver cancer in animal model.

博安霉素对小鼠结肠癌及其肝转移的抑制作用

目的 研究博安霉素对小鼠结肠癌及肝转移的疗效。方法 用小鼠结肠癌 2 6移植于皮下、盲肠、肝内及脾内移植肝转移等模型 ,并应用LEICAQ5 0 0IW图象分析系统分析肝转移病理切片。结果 博安霉素 5mg·kg-1和2 5mg·kg-1,对皮下瘤抑瘤率为 78 7%和 6 1 9% ;盲肠移植瘤抑瘤率为 99 4%和 90 0 % ;肝内移植瘤的抑瘤率为86 9%和 75 7%。博安霉素 10mg·kg-1和 5mg·kg-1对脾内原发灶抑瘤率为 98 1% ,99 4%和 5 2 6 % ,76 2 % ,对肝转移灶的抑瘤率为 93 1% ,97 6 %和 5 4 5 % ,5 6 8%。结论 博安霉素明显抑制小鼠结肠癌 2 6在皮下、盲肠、肝内和脾内移植瘤的生长 ,对肝转移也有显著抑制作用。

博安霉素药动学研究及临床评价

目的:研究博安霉素的药动学特点,并进行临床评价。方法:36例恶性肿瘤患者肌肉注射0.5～7.5mg/m2博安霉素后进行Ⅰ期临床试验,从中选择8例患者,采用微生物法进行药动学研究。结果:博安霉素对心、肝、肾、肺功能影响较小,无骨髓抑制作用;按5.29～7.65mg/m2剂量肌肉注射博安霉素后,t1/2α为(14.036±4.409)min.t1/2β为(39.160±5.599)min,AUC为(14.697±1.826)(μg·min)/ml,CLs为(0.781±0.102)L/min。结论:血清中博安霉素清除呈二室模型。

博安霉素诱导人鼻咽癌细胞凋亡作用

目的 :探讨博安霉素 (博莱霉素A6 )抗人鼻咽癌作用的机理。方法 :应用荧光显微镜、流式细胞仪及琼脂糖凝胶电泳等方法检测博安霉素诱导人鼻咽癌SUNE 1细胞凋亡的作用。结果 :当博安霉素浓度为 5 0 μg/ml,作用于SUNE 1细胞 10小时 ,荧光显微镜下SUNE 1细胞出现明显的凋亡形态学改变 ;流式细胞仪出现亚G1峰 ,凋亡率为 46 .3% ;琼脂糖凝胶电泳出现典型的DNA梯状条带。结论 :博安霉素具有体外诱导鼻咽癌细胞凋亡的作用 ,而诱导凋亡为其抗肿瘤作用的机制之一。

趋化肽fMLP增强博安霉素的抗肿瘤作用

背景和目的:博安霉素(Boanmycin,BAM)是博来霉素的A6组分,临床研究表明对多种肿瘤有效。趋化肽可吸引与激活白细胞(含巨噬细胞),使其对肿瘤生长、侵袭与转移过程产生一定的影响。本研究探讨BAM和趋化肽fMLP(N-formyl-methionyl-leucyl-phenylalanine,CHO-Met-Ile-Phe)联合抗肿瘤作用。方法:体外采用MTT法测定BAM和fMLP联合对肿瘤细胞的细胞毒作用,尤其是巨噬细胞存在时的作用。体内实验以小鼠移植性肝癌H22模型观察BAM和fMLP联合抗肿瘤作用。结果:体外实验,BAM与fMLP联合对肿瘤细胞没有协同作用,但BAM10μg/ml、30μg/ml和100μg/ml与fMLP20μg/ml联合在加入巨噬细胞后对肿瘤细胞有显著协同作用。体内实验时,fMLP1mg/mouse为瘤周给药×3,fMLP无显著抗肿瘤作用。BAM和fMLP合用时设计了3个实验:(1)肿瘤接种后24h开始治疗,BAM为瘤周给药×3,BAM0.5mg/kg在13天的抑瘤率为26.6%,合用fMLP后为64.7%,P<0.05,CDI值为0.36,有显著协同作用。(2)肿瘤接种后24h开始治疗,BAM为腹腔给药×3,BAM1mg/kg组肿瘤生长较快,合用fMLP后生长缓慢。在14天的抑瘤率为11%,合用fMLP后为70.6%,P<0.05,CDI为0.42,有显著协同作用。(3)肿瘤接种后96h开始治疗,BAM腹腔给药×3,BAM1mg/kg组肿瘤生长较快,合用fMLP后生长缓慢。在13天的抑瘤?

盐酸博安霉素临床药代动力学研究

采用“微生物法”，对我院８例癌症患者肌肉注射盐酸博安霉素进行血药浓度监测，其结果经３Ｐ８７程序进行药代动力学参数计算，结果表明：８例患者肌肉注射５．２９～７．６５ｍｇ／ｍ２盐酸博安霉素后，血清中的清除呈二室模型，平均药代动力学参数为：ｔ１／２ａ１４．０３６±４．４０９ｍｉｎ，ｔ１／２β３９．１６±５．５９９ｍｉｎ，ＡＵＣ１４．６９７±１．８２６μｇ／（ｍｌ·ｍｉｎ）。

国产一类抗癌新药盐酸博安霉素Ⅰ期临床研究

以３６例恶性肿瘤患者为观察对象，对国产一类抗癌新药盐酸博安霉素进行了Ｉ期临床研究。结果表明，主要毒副作用为发热、消化道反应、注射局部硬结，个别患者出现肌肉关节疼痛、皮疹、脱发。未见骨髓抑制作用，对心功能无影响，对肝、肾、肺功能影响较小，但可加重肾功能异常以及肺疾患、肺功能不全患者的肾、肺损伤。提出了Ⅱ期临床试用剂量，同时对６例患者进行了药代动力学研究。

国产一类抗癌新药盐酸博安霉素Ⅱ期临床研究

目的 对国产一类抗癌新药盐酸博安霉素（ＢＡＭ）治疗中、晚期恶性肿瘤的临床疗效和毒性进行评价。方法 对多中心应用ＢＡＭ 治疗中、晚期恶性肿瘤的效果进行Ⅱ期临床分析。全组３２５例，其中单用ＢＡＭ 治疗者１０５例，与其他药物联合治疗者２２０例。结果 单一用药的总有效率为３５％ ，对恶性淋巴瘤、头颈部癌和乳腺癌的有效率分别为６６７％ 、６５０％ 和３７５％ ；联合用药的总有效率为６４２％ ，对恶性淋巴瘤、乳腺癌、头颈部癌、食道癌和肺癌的有效率分别为８８５％ 、６２５％ 、５００％ 、４７２％ 和４５８％ 。盐酸博安霉素的主要不良反应有发热、寒战、肌肉疼痛、消化道反应、注射局部及皮肤反应，大多为Ⅰ～Ⅱ度。未见有骨髓抑制及肝肾功能损害。肺毒性显著低于博来霉素、培洛霉素及平阳霉素。结论 盐酸博安霉素对恶性淋巴瘤、头颈部癌、乳腺癌。

博安霉素抗人结肠癌作用的研究

对新抗肿瘤抗生素博安霉素 (BAM)在体外的抗人结肠癌作用、对癌细胞生物大分子合成、对相关癌基因和抗癌基因的表达及 DNA损伤和修复的影响进行了研究 ,采用细胞克隆形成法测定表明 ,博安霉素对人结肠癌 HT- 2 9细胞作用的 IC50 为 3.8× 10 - 8m ol/ L;NAG酶反应测定表明 ,博安霉素作用的 IC50 比其它博来霉素类药物低一个数量级。利用 [3 H]标记物掺入法测定表明 ,BAM对人结肠癌细胞的 DNA、RNA和蛋白质合成均有强抑制作用 ,其中对蛋白质合成的抑制作用最强。 Dot blot分析表明 ,博安霉素在 10 5mol/ L明显抑制人结肠癌细胞的 c- myc和 p5 3基因的表达 ,但增强 N- ras基因的表达。中性凝胶电泳分析表明博安霉素造成的人结肠癌细胞 DNA的损伤可被修复。

博安霉素抗人肝癌的作用及其作用机制的研究

目的 :研究新抗肿瘤抗生素博安霉素 (BAM )在体外的抗人肝癌的作用及其分子机制。方法 :采用细胞克隆形成法和NAG酶反应测定BAM抗肝癌作用 ,利用 [3H]标记物参入法测定BAM对肝癌细胞大分子合成的作用 ,利用斑点杂交检测BAM对肝癌细胞相关癌基因和抗癌基因表达的影响 ,利用中性凝胶电泳分析DNA损伤后的修复情况。结果 :BAM作用于BEL 740 2细胞克隆形成的最小IC50 为 8.6× 10 -9mol·L-1;对BEL 740 2细胞的杀伤的IC50 为 6 .2× 10 -6mol·L-1,表现出很强的抗肿瘤作用和良好的剂量 效应关系。对BEL 740 2细胞的DNA ,RNA和蛋白质合成均有强抑制作用 ,以抑制DNA和Pro合成的作用较强。在 0 .1～ 10 μmol·L-1浓度时 ,BAM明显抑制人肝癌细胞的c myc,N ras和p5 3基因的表达。BAM造成的人肝癌细胞DNA的损伤难以修复。 结论 :BAM是对人肝癌有效的抗肿瘤抗生素 ,其作用的分子机制与培洛霉素 (PEP)相似。

反相高效液相色谱法测定盐酸博安霉素含量

目的建立反相高效液相色谱法测定博安霉素含量的方法。方法 Agilent ZORBAX Eclipse PlusC18 （4.6mm ×150mm,5μm）色谱柱，流动相为0．005mol／L己烷磺酸钠离子对溶液（pH4．3）：甲醇：乙腈（72：21：7），柱温22℃，流速1mL／min，检测波长254nm。结果博安霉素在0．075-2．4mg／mL范围内，浓度与峰面积呈良好线性关系，相关系数R=0．9999。结论此方法操作简便、快捷、准确，重现性好，既可以用于博安霉素产品的质量分析，也可用于生产中间品的质量控制。与平阳霉素检测方法相比，采用此方法博安霉素与博莱霉素族其他组分的分离效果更好。

作用于基质金属蛋白酶的抗生素

基质金属蛋白酶 (matrix metalloproteinases,MMPs)是在与组织重塑有关的生理过程和以细胞外基质过度破坏为主要特征的病理过程中起重要作用的一类锌离子依赖性内肽酶。近些年来 ,MMPs在恶性肿瘤侵袭、生长和转移过程中的作用日益受到重视。我们以在肿瘤细胞破坏和穿过基底膜发生侵袭和转移过程中起重要作用的 MMPs家族成员 IV型胶原酶为研究对象 ,利用抗 IV型胶原酶单克隆抗体 3D6为工具 ,采用间接 EL ISA方法研究了四种抗生素对 IV型胶原酶或能够分泌 IV型胶原酶的 PG细胞的作用。结果表明 ,geldanamycin和博安霉素均能竞争性地抑制 3D6与 IV型胶原酶或 PG细胞的结合 ,说明其作用于 IV型胶原酶并可能对其活性产生抑制作用 ;而米诺环素在实验浓度下仅对 3D6和 PG细胞的结合表现出抑制作用。另外 ,我们发现地红霉素亦能够抑制单抗 3D6与 IV型胶原酶或 PG细胞的结合。结果表明 ,这四种抗生素可能作用于基质金属蛋白酶。

盐酸博安霉素脂质体的制备及表征

目的探讨制备方法对博安霉素脂质体包封率的影响,考察其制备的影响因素,优化处方工艺,并对其特性进行表征。方法分别采用乙醇注入法和逆向蒸发法制备博安霉素脂质体(boanmycin liposomes,BAM-Ls),确定最终工艺及处方。并对其包封率、粒径、Zeta电位和形态进行综合评价。结果逆向蒸发法制备BAM-Ls时药物包封率明显高于乙醇注入法,最终处方磷脂浓度为10mg/mL、磷脂胆固醇比为4:1(W/W)、水/乙醚比为1:3(V/V)时,所得脂质体包封率最高,约为30%。粒径为150nm,Zeta电位为-22.8mV,7d内脂质体稳定性良好。结论逆向蒸发法制备的BAM-Ls理化性质及稳定性良好。

博安霉素诱导人食管癌Eca-109细胞凋亡及其对细胞周期的影响

背景与目的:有研究报道博安霉素(boanmycin,BAM)是通过抑制肿瘤细胞的蛋白质合成来发挥抗人食管癌作用,但目前尚不清楚博安霉素是否还通过其他机制来实现其抗人食管癌作用。本文研究博安霉素诱导人食管癌Eca-109细胞凋亡及其对Eca-109细胞周期的影响。方法:运用形态学观察、流式细胞仪和琼脂糖凝胶电泳多种方法检测Eca-109细胞凋亡以及细胞周期分布的改变。结果:在经博安霉素处理的裸鼠移植瘤组织中,用电镜观察到不同阶段的凋亡细胞形态学改变;在体外实验中,博安霉素(50～125μg/ml)作用Eca-109细胞30～36h,可以诱导Eca-109细胞凋亡并出现DNA梯形条带,作用42h后将其细胞周期阻断于G2/M期。结论:博安霉素能诱导Eca-109细胞凋亡并将其细胞周期阻断于G2/M期。

抗癌新药博安霉素Ⅱ期临床研究

用盐酸博安霉素（ＢｏａｎｍｙｃｉｎＨｙｄｒｏｃｈｌｏｒｉｄｅ，ＢＡＭ）对８０例恶性肿瘤进行了Ⅱ期临床研究，单药治疗１５例，可评价疗效１３例，有效率７／１３（５３．８％）。其中舌癌３／４，下咽癌２／２，鼻腔癌１／１，口颊癌１／１。联合化疗６５例，可评价疗效６３例，总有效率３９／６３（６１．９％），其中恶性淋巴瘤１９／２０，肺癌９／１５，肝癌５／１７，鼻咽癌３／６，乳腺癌１／１，其它癌２／３，其主要毒性为发热或伴有轻度寒战。

盐酸博安霉素介入治疗肝癌103例

目的 :对盐酸博安霉素 (BAM )介入治疗原发性肝癌的临床疗效和毒性进行评价。方法 :入选 10 3例 ,单药组 6 5例 ,单用BAM ,肝动脉介入治疗 ,5 0mg ,共 2～ 3次 ;联合用药组 38例 ,每次除用BAM外加用 5 氟尿嘧啶(10 0 0mg)及丝裂霉素 (10～ 2 0mg) ,共 2～ 3次。结果 :单药组有效率 30 .8% ,联合用药组有效率 4 2 .1%。BAM的主要不良反应有发热、寒战、恶心、呕吐。未见骨髓抑制及心、肝、肾功能损害 ,无明显肺毒性反应。结论 :BAM是原发性肝癌介入治疗的有效药物。